1. Field of the Invention
This invention concerns a method of protecting proteins such that their biological activity is preserved after freezing. This protection is achieved by adding a cryoprotectant that preserves biological activity of the protein in spite of freezing or lyophilization. More particularly, the invention concerns a method for preserving therapeutically effective proteins so that they can be conveniently stored in a nonliquid form while retaining substantially all of their biological activity.
2. General Discussion of the Background
Proteins are one of the almost universal constituents of living organisms. The fragility of living organisms and their usual ability to survive only within very narrow ranges of environmental conditions can be explained by a protein's loss of biological activity outside of a relatively narrow temperature range. For example, freezing often permanently changes the three dimensional tertiary structure of proteins, usually resulting in a loss of biological activity.
An especially sensitive kind of protein is an enzyme. Enzymes are polypeptide molecues that are produced by living cells and catalyze specific biochemical reactions at body temperatures. An example of such an enzyme is phosphofructokinase (PFK) which is a rate limiting catalyst in the glycolytic pathway. PFK catalyzes the addition of a phosphate group to fructose 6-phosphate, but once the enzyme is frozen it irreversibly loses all catalytic activity.
Since proteins play an important role in the function and regulation of living organisms, proteins have also become useful pharmaceutical agents. For example, the pancreatic protein insulin is instrumental in controlling animal blood sugar levels. If an animal's production of insulin is impaired, the resulting physiological condition is known as diabetes. This disease is usually treated by injecting specific doses of insulin into the animal. The cost and inconvenience of such treatment is increased, however, by the necessity of refrigerating the insulin in liquid form to preserve its biological activity until it is administered to a patient. Even at refrigeration temperatures, however, the protein is unstable and loses some of its activity. It would therefore be desirable to freeze such proteins to give them a longer shelf life.
It is not presently possible to simply freeze a protein such as insulin because freezing and subsequent thawing usually diminishes the biological activity of the protein. This problem has resulted in efforts to find ways to preserve the biological activity of proteins after they are frozen or lyophilized.
For example, U.S. Pat. No. 4,180,917 discloses a multistep process for freeze-drying enzymes in which the enzymes typically recover about 75 to 85% of their biological activity following freeze-drying. The method is complicated by the need for concentrating the enzyme solution using reverse osmosis or ultrafiltration and adding water insoluble salts. The protease and alpha-amylase compositions treated by this method are also quite stable to begin with and would retain a substantial amount of their activity after freezing even without addition of insoluble salts and reverse osmosis.
U.S. Pat. No. 3,607,858 describes a method of freeze-drying human blood proteins in the presence of small amounts of nonionic surface active agents with very rapid freezing in small containers. The globulins treated by this method are already quite stable to freezing and would survive freeze-drying even without addition of surface active agent. The addition of the nonionic surfactant simply serves to speed up the process of redissolving the globulin.
Although not dealing with preservation of proteins, U.S. Pat. No. 4,134,214 discloses that a polysaccharide antigen can be preserved by freeze-drying it at temperatures of -20.degree. to -40.degree. C.
Similarly unrelated to protein preservation is the work of Crowe et al. at the University of California-Davis with liposomes. A liposome is an artificial vesicle comprised of one or more concentric phospholipid bilayers. Crowe has shown, for example, in Science, vol. 223, pp. 701-703 (Feb. 17, 1984), that addition of trehalose alone to liposomes allows them to be freeze-dried and rehydrated without disruption of their phospholipid membranes. The mechanism of protection suggested by Crowe is a direct interaction between the phospholipid polar head groups and trehalose that prevents adhesion between the head groups during freezing. Crowe also believes that trehalose reduces the transition temperature of the liposomes and inhibits thermotropic gel to liquid crystalline phase transitions that are known to result in leakage of the contents of hydrated phospholipid vesicles.
The prior art had suggested that cryoprotectants such as dimethylsulfoxide (DMSO) and glycerol exerted protective action on proteins by altering the structure of the water solvent through a thermodynamic effect. Gekko et al., Biochemistry 20:4667-4676 (1981). It would therefore not be predicted that trehalose would protect proteins since Crowe had taught that the mechanism of trehalose action was one of direct interaction with the substance being protected.
The use of prior cryoprotectants, such as DMSO, with proteins present serious problems since DMSO and other cryoprotectants are biologically incompatible materials. If such an incompatible material were added to proteins, the cryoprotectant had to be removed prior to use of the protein in a biological system to prevent toxic reactions. If the incompatible material had reducing properties, it could also cause "protein browning" which diminishes or destroys the activity of the protein and turns it brown. This protein browning phenomenon is discussed in Lea, C. H. and R. S. Hannan, Biochim. Biophys. Acta., 3:313 (1949); Lea, C. H. and R. S. Hannan, Biochim. Biophys. Acta., 4:518 (1950); Lea, C. H. and R. S. Hannan, Biochim. Biophys. Acta., 5:433 (1950); Lea, C. H. and R. S. Hannan, Nature, 165:438 (1950); Feeney, R. E., G. Blankenhorn and H. Dixon, Adv. Prot. Chem., 29:135 (1975).
Another problem with prior art cryoprotectants such as DMSO and glycerol is that they must be present in solution in several molar amounts before they exert their cryoprotective influence. Such excessive amounts of an additive can disrupt biological function and are difficult to remove.
It is accordingly an object of this invention to provide a method of protecting proteins such that their biological activity is preserved after freezing.
Another object of the invention is to provide such a method of preservation which will protect the protein during freezing and thawing or during lyophilization and subsequent rehydration.
Yet another object of the invention is to provide such a method of preservation which employs only nontoxic, biologically compatible additives.
Still another object of the invention is to provide such a method that will permit proteins, such as therapeutically useful substances and enzymes, to be frozen and thawed or lyophilized and rehydrated while retaining the majority of their biological activity.
Even yet another object is to provide a cryogenic protectant additive for proteins which is nonreducing and does not cause protein browning.
Finally, it is an object to provide such a method which employs only a very low concentration of a cryoprotectant additive which is nontoxic and can be administered to an animal along with a therapeutic protein.